NGS Expert Blog

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Newts have an extraordinary ability to regenerate tissues. For example, they can re-grow fully functional limbs after amputation. In addition, regeneration of parts of the central nervous system, the heart, and the lens has been characterized, making them an excellent model organism for studying regenerative processes. However, because of their enormous genome size (10 times that of human), the molecular mechanisms behind this amazing regenerative process are largely unknown.

A research group at the Max Plank Institute recently published a de novo assembly of the transcriptome of the urodelian amphibian Notophthalmus viridescens (Looso M. et al. ). The researchers combined 454, Illumina, and Sanger sequencing data from both normalized and non-normalized cDNA libraries. The resulted transcriptome comprises over 120,000 non-redundant transcripts. Homology search using BLAST led to annotation of 38,000 transcripts. Importantly, they found 800 transcripts, whose protein-coding potential was validated by mass spectrometry, that show no similarity to any know transcripts or show similarity to urodele-specific EST sequences. Some of these transcripts belong to novel protein families.

It is an interesting hypothesis that some of those newt-specific proteins may provide mechanistic insights into regeneration processes unique to these animals. Their work will definitely be an important resource for subsequent studies in tissue regeneration and may benefit future research in regenerative medicine.

With today’s press release I am happy to announce that AROS Applied Biotechnolgy A/S  is now a member of the Eurofins group.

Here is a short introduction of our new colleagues from AROS:

• AROS was founded in the year 2000
• AROS started as a spin off of from the Aarhus University Hospital and was the first service provider for Affymetrix in Europe
• AROS is based in Denmark and provides a long term experience in sample preparation, microarray analysis and next generation sequencing (NGS)
• Nowadays AROS has a leading position in NGS service for pharmaceutical research
• AROS is an Illumina reference lab for next generation sequencing
• The main focus in NGS is RNA-Seq and exome sequencing that is accomplished with the exome designs of the leading provider in this area (Illumina TruSeq Exome Enrichment, NimbleGen EZ Capture & Agilent SureSelect)

“AROS is an excellent fit […] with our focus on high-quality next-generation sequencing […]” (Dr. Gilles Martin) and therefore I am confident that this new alliance will help us both in further expanding our experience level in NGS and to benefit from our complementary strength.

I am sure you will hear more about the activities from AROS on our blog and hope you join me in welcoming AROS as a member of Eurofins.

Opinions differ as to whether next generation sequencing is already mature enough to be a useful tool in diagnostic routine.

Below you can find an interview of the cardiologists from the university of Heidelberg about their studies to integrate next generation sequencing into a diagnostic tool. Therefore they do collaborate with Siemens to receive best possible results that can be used by the doctors in the same way as current reports from other technologies.

Discover 2270 high-throughput sequencing machines in 830 centres at http://www.omicsmaps.com/. Select according to a specific platform or search for a facility or a region of your interest. Relevant statistics are also available at http://www.omicsmaps.com/stats.

No matter if you are a commercial service provider, a researcher or a device manufacturer, you will love the map!

Dear all,

Here is my second summary from 4th NGS Congress at London Heathrow end of 2012. It will bring to you some (hopefully) interesting new facts about sequencing with PacBio RS - the second long read technology present in the actual markets and also the only system delivering reads even longer than 10,000 bp…

Kevin Corcoran, Senior Vice President at Pacific Biosciences held an interesting and very nice talk about the most recent developments for the PacBio RS system. He also showed some nice detailed road maps about future aims and plans. One important thing actual to be mentioned is the launch of the new “XL Chemistry” – while still “C2 Chemistry” may be used as well. The other very interesting story is about “Stage Start” a new feature enabling a parallel start of all sequencing detection similar to the well-known “hot start” technology for PCR. Such detection of sequences better will start from a defined position for most of the libraries than starting from somewhere in the middle. Last but not least, I’m very keen to learn how the future “Photo Protected DNA Polymerases” may further develop – an idea being really very, very next-next-generation…

First of all I can summarize that applying “XL Chemistry” looks really interesting and this being true also in terms of Eurofins MWG Operon de novo sequencing and assembly focus.  This new feature of the PacBio RS machine may also open some new doors to other types of applications, while in general the need for extrem high data coverages may be reduced in parallel.

Currently “C2 Chemistry” is on the machine and running a 90 min video may deliver you about 20-50,000 reads and data outputs of 30-50 Mb – of course higher yields may be possible for “ideal” DNA samples. The average read length is about 3,000 bp (!), while the 95% percentile is about 8,000bp. With the new ”XL Chemistry” we got an average yield of about 40,000 reads per SMRT cell with an average read length of about 4,000bp (+30%). Overall, we are very pleased with these first results, especially since we see some good potential to further increase data yields using the new software pipeline started in parallel (Hierarchical Genome Assembly Process and Quiver).

— See picture 1: —

It is also important to mention two different ways of “How-to-deal” with the XL Chemistry. 1) ”XL chemistry for Polymerase binding”, but “C2 chemistry for sequencing”. This allows for longer reads at the same quality (currently we still do have a single error rate of 10% to 20 %, average maybe 15%). 2) “XL chemistry for Polymerase binding” AND ”XL chemistry for sequencing”. Such one can yield even longer reads, but unfortunately the error rate will also increase by a few %. Therefore this method is being recommended especially for de novo assembly or finishing genomes.

— See picture 2: —

Finally one real “next-next-gen” highlight was the presentation of a development at Pacific Biosciences scoping with the idea to protect the polymerase enzyme from being killed by the energy of the laser. A picture shows how this should work in principle - by setting in place a laser-light protecting sun-blocker - this story was really fascinating for me and I hope to see in future more than the very promising first data results …

— See picture 3: —

So over all Pacific BioSciences keeps also moving very fast in year 2013 and it will be very nice to see and learn how all these additional improvements and new features may  improve the overall data results of this fascinating very long read technology offering today real single reads longer than 10,000 bp.

Cheers now and see you on our next BLOG,
Axel

Domestication of goats happened already thousands of years ago. Nowadays they are also used as models for biomedical research. However, one thing was still missing: a reference genome. Researchers from China could now close this gap by successfully sequencing the genome of a domestic goat.

To reveal the secrets of the goat genome the researchers applied a hybrid approach of Illumina shotgun sequencing and whole genome mapping (WGM) using the Argus system from Opgen. As a result, the number of scaffolds could be reduced from 2,090 to 315. This demonstrates that whole-genome mapping for large genomes can be a replacement for traditional genetic maps for de novo assembly (Dong et. al).

This reference genome can now be used for mapping reads of other goats to identify SNPs and other variants that could play a role for breeding, cashmere fiber prodcution or different goat behaviours (Dong et. al).

If you are interested in more information about optical mapping, read our dedicated blog posts: What is optical mapping? and Creating the perfect genome assembly.

Researchers of the Wellcome Trust Sanger Institute have reported DNA sequencing on the PacBio RS sequencer without prior library preparation. As described in an article in BioTechniques last month, the method has so far been applied for sequencing single- and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA modification analysis, as well as linear DNA fragments covering an entire bacterial genome.

The standard library preparation step was skipped and the DNA was directly used in the sequencing reaction. With this approach, the researchers around first author Paul Coupland were able to generate sequencing data with as little as 1 ng of starting material, taking only about 8 hours of time.

“In terms of read length and accuracy, the direct sequencing method is comparable to the standard sequencing protocol on the PacBio”, as Coupland told InSequence. “There are no drawbacks in terms of read length and accuracy because PacBio is already single molecule sequencing, so it’s just skipping the library prep and going straight into the sequencing part.”

Since random hexamers can be applied as sequencing primers, and no growth of organisms is needed during sample preparation, the method can be applied without any a priori information on the organisms in the sample.

Clearly, this technique still needs to be optimised. For example, the sequence yield obtained with this approach is considerably lower than with standard methods (3,000 reads per SMRT cell, in contrast to 35,000 to 50,000 reads for standard methods).

However, the authors think that the technique has great potential for clinical applications, where unknown organisms need to be quickly identified. As Dr. Harold Swerdlow, lead author from the Wellcome Trust Sanger Institute says in their press release: “Our technique can be performed without any prior knowledge of the sequence and with no organism specific reagents, in a short space of time. This makes it a promising alternative for clinical situations such as infection control.”

Watch out the presentation of the SITN Boston talking about whole genome sequencing and its impact on personalised medicine.

Further recorded lectures given by graduate students at Harvard and focusing on hot topics in science research and news can be found at https://sitn.hms.harvard.edu/seminar-archive-2012/. Enjoy!

The analyst and sequencing community is currently divided on whether to believe the rumors of a new bid from Roche to buy Illumina. The source of the controversial discussions is an article from the Swiss Newspaper L’Agefi that reported end of December that Roche and Illumina might have agreed to a deal for Roche to acquire Illumina. Since Illumina turned down Roche’s original bid in January, continuous interest from Roche has been reported several times, but the report from L’Agefi is also mentioning concrete amounts of the bid. According to them, the acquisition might take place for $66 per share, valuing the deal at about $8.14 billion in total.

The offer is $15 per share higher than the previous offer of $51 in April last year. According to the analyst Devia Ferreiro of Oppenheimer the new bid is definitely at a level that might lead to a final deal.

With Roche having only about 9% of the NGS market and next generation sequencing becoming most likely an important clinical diagnostic tool in the next years, the strategy focus of Roche must be to get better access to the NGS market and to take NGS to clinical practice. The acquisition of the NGS market leader Illumina represents an optimal starting point.

We’ll see if the rumors are built on a solid foundation within the next two weeks: The Swiss newspaper L’Agefi reported that the announcement might come during the first half of January.